Glycolytic activation of monocytes regulates the accumulation and function of neutrophils in human hepatocellular carcinoma.

BACKGROUND & AIMS: Neutrophils are one of the most abundant components in human hepatocellular carcinoma (HCC) and have been shown to play important roles in regulating disease progression. However, neutrophils are very short-lived cells in circulation, and mechanisms regulating their accumulation and functions in HCC are not yet fully understood. METHODS: Monocytes were purified from non-tumor or paired tumor tissues of patients with HCC, and their production of neutrophil-attracting chemokines was evaluated. Mechanisms regulating the expression of CXCL2/8 by tumor monocytes, and the role of tumor monocyte-derived chemokines and cytokines in modulating neutrophil accumulation and functions were studied with both ex vivo analyses and in vitro experiments. RESULTS: Monocyte-derived CXCL2 and CXCL8 were major factors in regulating the recruitment of neutrophils into tumor milieus. These chemokines, in addition to tumor-derived soluble factors, could inhibit apoptosis and sustain survival of neutrophils, thus leading to neutrophil accumulation in tumor tissues. Moreover, monocyte-derived TNF-α acted synergistically with tumor-derived soluble factors to induce the production of the pro-metastasis factor OSM by neutrophils. Further, the glycolytic switch in tumor-infiltrating monocytes mediated their production of CXCL2 and CXCL8 via the PFKFB3-NF-κB signaling pathway. Accordingly, levels of PFKFB3, CXCL2/CXCL8 production in monocytes and infiltration of OSM-producing neutrophils were positively correlated in human HCC tissues. CONCLUSIONS: Our results unveiled a previously unappreciated link between monocytes and neutrophils in human HCC, identifying possible targets that could be therapeutically exploited in the future. LAY SUMMARY: Neutrophils constitute a major but poorly understood component of human hepatocellular carcinoma (HCC). Herein, we unveil a novel mechanism by which metabolic switching in monocytes promotes the accumulation of neutrophils in the tumors of patients with HCC. Both monocyte-produced chemokines and signals from the tumor microenvironment promote the production of the pro-metastatic factor OSM by neutrophils. These data identify potential targets for immune-based anticancer therapies for HCC.

Glycolytic activation of peritumoral monocytes fosters immune privilege via the PFKFB3-PD-L1 axis in human hepatocellular carcinoma.

BACKGROUND & AIMS: Programmed cell death 1 ligand 1 (PD-L1) expression on antigen-presenting cells is essential for T cell impairment, and PD-L1-expressing macrophages may mechanistically shape and therapeutically predict the clinical efficacy of PD-L1 or programmed cell death 1 blockade. We aimed to elucidate the mechanisms underlying PD-L1 upregulation in human tumor microenvironments, which remain poorly understood despite the clinical success of immune checkpoint inhibitors. METHODS: Monocytes/macrophages were purified from peripheral blood, non-tumor, or paired tumor tissues of patients with hepatocellular carcinoma (HCC), and their possible glycolytic switch was evaluated. The underlying regulatory mechanisms and clinical significance of metabolic switching were studied with both ex vivo analyses and in vitro experiments. RESULTS: We found that monocytes significantly enhanced the levels of glycolysis at the peritumoral region of human HCC. The activation of glycolysis induced PD-L1 expression on these cells and subsequently attenuated cytotoxic T lymphocyte responses in tumor tissues. Mechanistically, tumor-derived soluble factors, including hyaluronan fragments, induced the upregulation of a key glycolytic enzyme, PFKFB3, in tumor-associated monocytes. This enzyme not only modulated the cellular metabolic switch but also mediated the increased expression of PD-L1 by activating the nuclear factor kappa B signaling pathway in these cells. Consistently, the levels of PFKFB3+CD68+ cell infiltration in peritumoral tissues were negatively correlated with overall survival and could serve as an independent prognostic factor for survival in patients with HCC. CONCLUSIONS: Our results reveal a mechanism by which the cellular metabolic switch regulates the pro-tumor functions of monocytes in a specific human tumor microenvironment. PFKFB3 in both cancer cells and tumor-associated monocytes is a potential therapeutic target in human HCC. LAY SUMMARY: Programmed cell death 1 ligand 1 (PD-L1) expressed on antigen-presenting cells, rather than tumor cells, has been reported to play an essential role in checkpoint blockade therapy. A fundamental understanding of mechanisms that regulate the expression of PD-L1 on tumor-infiltrating monocytes/macrophages will undoubtedly lead to the possibility of developing novel PD-L1 blockade strategies with high specificity and efficiency. The current study unveils a novel mechanism by which metabolic switching links immune activation responses to immune tolerance in the tumor milieu, identifying potential targets for future immune-based anti-cancer therapies.

Downregulation of phosphoserine phosphatase potentiates tumor immune environments to enhance immune checkpoint blockade therapy.

BACKGROUND: Effects of immune checkpoint blockade (ICB) treatment in hepatocellular carcinoma (HCC) are limited. The current study explored the possibility of exploiting tumor metabolic switches to enhance HCC sensitivity to immune therapies. METHODS: Levels of one-carbon (1C) metabolism and the expression of phosphoserine phosphatase (PSPH), an upstream enzyme of 1C pathway, were evaluated in paired non-tumor and tumor tissues from HCC. Underlying mechanisms mediating the role of PSPH in regulating the infiltration of monocytes/macrophages and CD8+ T lymphocytes were studied through both in vitro and in vivo experiments. RESULTS: PSPH was significantly upregulated in tumor tissues of HCC and its levels were positively correlated with disease progression. PSPH knockdown inhibited tumor growth in immunocompetent mice, but not in those with macrophage or T lymphocyte deficiencies, indicating the pro-tumor effects of PSPH were dependent on both immune components. Mechanistically, PSPH facilitated monocytes/macrophages infiltration by inducing the production of C-C motif chemokine 2 (CCL2), while at the same time reduced CD8+ T lymphocytes recruitment through inhibiting the production of C-X-C Motif Chemokine 10 (CXCL10) in tumor necrosis factor alpha (TNF-α)-conditioned cancer cells. Glutathione and S-adenosyl-methionine were partially involved in regulating the production of CCL2 and CXCL10, respectively. shPSPH (short hairpin RNA) transfection of cancer cells enhanced tumor sensitivity to anti-programmed cell death protein 1 (PD-1) therapy in vivo, and interestingly, metformin could inhibit PSPH expression in cancer cells and mimic the effects of shPSPH in sensitizing tumors to anti-PD-1 treatment. CONCLUSIONS: By tilting the immune balance towards a tumor-friendly composition, PSPH might be useful both as a marker in stratifying patients for ICB therapy, and as an attractive therapeutic target in the treatment of human HCC.

The Effects of Hedgehog Signaling Pathway on the Proliferation and Apoptosis of Melanoma Cells.

BACKGROUND: Studies have found that the abnormality of the Hedgehog signaling pathway is related to the occurrence and development of a variety of tumors, but the effect of this signaling pathway on melanoma cells is still unclear. METHODS: This study aimed to discuss the effect of Hedgehog signaling pathway on the proliferation and apoptosis of human malignant melanoma A375 cells and explore its possible mechanism in the proliferation and apoptosis of melanoma cells. Different concentrations of Hedgehog signaling pathway inhibitor cyclopamine (5, 10, 20 and 40 µM) were used to treat human melanoma A375 cells for 24, 48, and 72 h, and set a blank control group (0 µM). Trypan blue cell counting method was used to detect cell viability. MTT method was used to detect the inhibition rate of cell proliferation. Transwell was used to detect cell invasion, and flow cytometry was used to detect cell apoptosis. RESULTS: Through the trypan blue cell counting method and MTT experiment, it was found that the Hedgehog signaling pathway inhibitor cyclopamine has an inhibitory effect on the proliferation and viability of melanoma A375 cells (P < 0.05), and the proliferation inhibitory effect is enhanced with prolonged action time in a dose- and time-dependent manner. Transwell experiment showed that compared with the blank control group, the invasion and migration ability of the treated melanoma A375 cells are significantly reduced, and the difference is statistically significant (P < 0.05). Cell apoptosis experiment showed that compared with the blank control group, the apoptosis rate of A375 cells is significantly higher after treated by 40 µM cyclopamine for 24 h, and the difference is statistically significant (P < 0.05). Gli1 and Bcl-2 protein are highly expressed in melanoma A375 cells, and their expressions show a downward trend (P < 0.05) after being treated by cyclopamine. CONCLUSION: Cyclopamine inhibits cell proliferation and induces cell apoptosis by downregulating Gli1. Hedgehog signaling pathway can be used as a new target for the treatment of malignant melanoma, and multiple measures can be used to inhibit the signaling pathway to achieve a therapeutic effect.

Carbonic anhydrase XII mediates the survival and prometastatic functions of macrophages in human hepatocellular carcinoma.

Macrophages constitute a major immune component in tumor tissues, but how these cells adapt to and survive in the nutrient-depleted and lactic acid-induced acidic tumor microenvironments is not yet fully understood. Here, we found that levels of carbonic anhydrase XII (CA12) expression were significantly and selectively upregulated on macrophages in human hepatocellular carcinoma (HCC). Transient glycolytic activation of peritumoral monocytes induced sustained expression of CA12 on tumor-infiltrating macrophages via autocrine cytokines and HIF1α pathways. On the one hand, CA12 mediated the survival of macrophages in relatively acidic tumor microenvironments, while on the other hand, it induced macrophage production of large amounts of C-C motif chemokine ligand 8 (CCL8), which enhanced cancer cell epithelial-mesenchymal transition (EMT) and facilitated tumor metastasis. Consistently, the accumulation of CA12+ macrophages in tumor tissues was associated with increased tumor metastatic potential and reduced survival of patients with HCC. Selective targeting of tumor-infiltrating macrophages with a CA12 inhibitor reduced tumor growth in mice and was sufficient to synergistically enhance the therapeutic efficacy of immune-checkpoint blockade. We suggest that CA12 activity is a previously unappreciated mechanism regulating the accumulation and functions of macrophages in tumor microenvironments and therefore represents a selective vulnerability that could be exploited in future designs for antitumor immunotherapeutic strategies.

Neutrophil extracellular traps induce tumor metastasis through dual effects on cancer and endothelial cells.

Neutrophils constitute a major component in human hepatocellular carcinoma (HCC) and can facilitate disease progression via poorly understood mechanisms. Here, we show that neutrophil extracellular traps (NETs) formation was increased in human HCC tumor tissues than in paired non-tumor liver tissues. Mechanism study revealed that tumor-induced metabolic switch toward glycolysis and pentose phosphate pathway in tumor infiltrating neutrophils promoted NETs formation in a reactive oxygen species dependent-manner. NETs subsequently induced the migration of cancer cells and down-regulation of tight junction molecules on adjacent endothelial cells, thus facilitating tumor intravasation and metastasis. Accordingly, NETs depletion could inhibit tumor metastasis in mice in vivo, and the infiltration levels of NETs-releasing neutrophils were negatively associated with patient survival and positively correlated with tumor metastasis potential of HCC patients. Our results unveiled a pro-metastatic role of NETs in the milieu of human HCC, and pointed to the importance of metabolic reprogramming in shaping their characteristics, thus providing an applicable efficient target for anti-cancer therapies.

Sedative and Hypnotic Effects and Transcriptome Analysis of Polygala tenuifolia in Aged Insomnia Rats.

OBJECTIVE: To study the sedative and hypnotic effects and underlying mechanisms of Polygala tenuifolia (PT) on treating aged insomnia rats. METHODS: Sixty Sprague-Dawley male rats were divided into 6 groups by a random number table, including control group, model group, diazepam group (0.92 mg/kg), as well as PT low-, medium- and high-dose groups (0.0875, 0.175, 0.35 g/kg, respectively), 10 rats in each group. Aged insomnia rat model was established with subcutaneous injection of D-galactose for 42 days and then intraperitoneal injection of para-chlorophenylalanine for 3 days. PT and diazepam were respectively given to aged insomnia rats by intragastric administration for 7 days after model establishment. Then the rats were investigated by body weight, Morris water maze test, pentobarbital test, enzyme-linked immunosorbent assay, and transcriptome sequencing. RESULTS: Compared with the model group, PT increased the body weight, improved memory ability, and prolonged pentobarbital-induced sleep time of aged insomnia rats (P<0.01 or P<0.05). The medium dose of PT also increased the neurotransmitter levels of 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA), and decreased the level of Glu in the hippocampus of aged insomnia rats (P<0.05 or P<0.01). Twenty-four differentially expressed genes (DEGs) were overlapped among model group, medium-dose PT group, and diazepam group in transcriptome analysis. Fuom and Pcp2 were down-regulated by the treatment of medium-dose PT (P<0.01 or P<0.05). The metabolic pathways of PT were relatively less than diazepam (91 vs. 104). CONCLUSIONS: The sedative and hypnotic effects of PT in aged insomnia rats might be related to neuro, metabolism pathways, especially through GABAergic signaling pathway. It provided more effective herb choice for the treatment of senile insomnia.